Linalool attenuated ischemic injury in PC12 cells through inhibition of caspase‐3 and caspase‐9 during apoptosis

Abstract Numerous studies have indicated the pharmacological properties of linalool, a volatile terpene alcohol found in many flowers and spice plants, including anti‐nociceptive, anti‐inflammatory, and neuroprotective activities. The aim of this study was to explore the mechanisms of neuroprotection provided by (±) linalool and its enantiomer, (R)‐(−) linalool against oxygen, and glucose deprivation/reoxygenation (OGD/R) in PC12 cells. PC12 cells were treated with (±) linalool and (R)‐(−) linalool before exposure to OGD/R condition. Cell viability, reactive oxygen species (ROS) production, malondialdehyde (MDA) level, DNA damage, and the levels of proteins related to apoptosis were evaluated using MTT, comet assay, and western blot analysis, respectively. IC50 values for the PC12 cells incubated with (±) linalool and (R)‐(−) linalool were 2700 and 2600 μM after 14 h, as well as 5440 and 3040 μM after 18 h, respectively. Survival of the ischemic cells pre‐incubated with (±) linalool and (R)‐(−) linalool (100 μM of both) increased compared to the cells subjected to the OGD/R alone (p < .001). ROS and MDA formation were also decreased following incubation with (±) linalool and (R)‐(−) linalool compared to the OGD/R group (p < .01). In the same way, pre‐treatment with (±) linalool and (R)‐(−) linalool significantly reduced OGD/R‐induced DNA injury compared to that seen in OGD/R group (p < .001). (±) Linalool and (R)‐(−) linalool also restored Bax/Bcl‐2 ratio and cleaved caspase‐3 and caspase‐9 (p < .001, p < .01) following ischemic injury. The neuroprotective effect of linalool against ischemic insult might be mediated by alleviation of oxidative stress and apoptosis.


| INTRODUC TI ON
Lack of glucose and oxygen supply to an area of brain due to decreased blood flow to the brain's tissue causes ischemic strokes, the most prevalence form of strokes (Chiu et al., 2014).
Considering high percentage of mortality and disability due to stroke (Chen et al., 2017), as well as limitation in the current treatments, recent studies focus on promising strategies for prevention and treatment of ischemic cerebral injury (Page et al., 2012).
A number of critical pathological processes including excitotoxicity, oxidative stress, and inflammation have been proposed to be involved in the cerebral ischemia (Lee et al., 2014). These alterations trigger death signaling pathways inducing cell apoptosis (Rodrigo et al., 2013).
Linalool has been shown to reduce the neuropathic pain via interaction with NMDA (N-methyl-D-aspartate) receptors and suppression of the pro-inflammatory cytokines (Peana et al., 2004;Pereira et al., 2018). Also, the anti-depression and anxiolytic effects have been mediated through modulating monoaminergic system and antagonizing NMDA receptors (Pereira et al., 2018). Linalool also alleviated learning and spatial memory deficits, reduced βamyloid plaques, tauopathy, and pro-inflammatory mediators (p38 MAPK, NOS2, COX2, and IL-1b) in the hippocampus and amygdala of D-galactose and aluminum trichloride-induced Alzheimer's disease mouse model (Xu et al., 2017a). Linalool was found to induce neuroprotective effects through suppression of ROS, restoring antioxidant enzymes, as well as inhibition of microglia migration (Li et al., 2015). Moreover, linalool could protect neuronal cells from apoptotic cell death following ischemic/hypoxic injury (Park et al., 2016). Activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) pathway by (R)-(−) linalool could protect against oxidative injury (Li et al., 2015;Xu et al., 2017aXu et al., , 2017b. This study was aimed to evaluate the neuroprotective and antiapoptotic effects of both (R)-(−) linalool and (±) linalool in PC12 cells subjected to oxygen and glucose deprivation/reoxygenation (OGD/R), as a model of cerebral ischemia-reperfusion injury.

| Cell culture and treatments
The PC12 cells were cultured in high glucose DMEM (containing 4500 mg/L of glucose) which was supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin) in a chamber with a humidified atmosphere of 5% CO 2 , at 37 °C. The cells were cultured in a 96-well or 6-well culture plates (at a density of 10 4 and 10 6 cell/well, respectively). Thereafter, the culture media of each well were replaced with fresh media containing 1, 1.5, 3, 6, 12.5, 25, 50, 100, 200, 800, 1600, 2000, 2500, and 3200 μM of (R)-(−) linalool and (±) linalool. After 2 or 6 h pre-treatment with linalool, the cells were exposed to OGD/R condition (an in vitro model of cerebral ischemia and reperfusion injury).
To produce OGD/R condition, the PC12 cell were placed in an anaerobic incubator (containing 1% O 2 , 94% N 2 , and 5% CO 2 ) with the glucose-free DMEM medium at 37°C. After 12 h, the cells were then returned to a normoxic incubator (95% air and 5% CO 2 ) with normal glucose medium for 12 h. Finally, the cells were harvested for the following experiments (Forouzanfar et al., 2021).

| Cell viability determination
At the end of treatment period, PC12 cell viability was determined using the MTT colorimetric method (Sadeghnia et al., 2014). One hundred microliters of MTT solution (5 mg/mL) was transferred to each well and incubated for 3 h at 37°C. After adding DMSO (150 μl), the absorbance of soluble formazan was measured with a microplate reader at 450-630 nm.
In addition, the half maximal inhibitory concentrations (IC 50 ) of (R)-(−) linalool and (±) linalool were estimated according to the viability of PC12 cells.

| Determination of MDA formation
Malondialdehyde production, a marker of lipid peroxidation, was measured based on thiobarbituric acid-reactive substances, as previously described (Hamid Reza Sadeghnia et al., 2017). At the end of treatments, a cell lysate was prepared by scraping the cell monolayers with trichloroacetic solution (1 ml, 2.5%). The homogenate was centrifuged (4000 g, 10 min, 4°C) and thiobarbituric acid (0.67% w/v, 0.8 ml) and trichloroacetic acid (15% 2 w/v, 0.4 ml) reagents were mixed with the supernatant. The samples were then placed in a boiling water bath. After cooling and centrifugation, the absorbances of supernatants were recorded by plate reader.
Bradford assay was performed to calculate the protein concentration in the cellular extracts and the MDA concentration was presented as nmol/mg protein based on a standard curve of MDA solution.

| Comet assay
When the treatments of the cells, with or without linalool, were finished, the cells were harvested and suspended in low melting point agarose (1%, LMP in PBS). After that, they were placed on the glass slides, which were pre-coated with normal melting agarose (1%, NMP in PBS). After the gel solidification of a third layer of LPM, the slides were exposed to lysis buffer at 4°C overnight. DNA unwinding was performed using an alkaline solution (300 mM NaOH, 1 mM Na 2 EDTA, pH > 13). After electrophoresis at 23 V and 300 mA at 4°C for 30 min, and removing the excess alkali and detergents (by washing with tris buffer, 0.4 M, pH 7.5), and staining with edithium bromide, the stained slides were examined by a fluorescence microscope (excitation filter 520-550 nm and barrier filter 580 nm) and photographed (Rajabian et al., 2016).

| Western blot analysis of Bax, Bcl-2, caspase-3, and cacspase-9 expressions
After the treatments, the total proteins were extracted by ice-cold lysis buffer and with the addition of protease inhibitor. Equal amount of protein samples was loaded on SDS-PAGE, fractionated and then transferred to PVDF. The membranes were blocked in non-fat milk-TBS (5% w/v), and then exposed to the indicated primary and HRP-conjugated secondary antibodies, and visualized by chemiluminescence using an ECL reagent. Band densities were quantified by NIH Image J software (Rajabian et al., 2019).

| Statistical analyses
All statistical analyses were carried out using the GraphPad Prism, version 8. All data were expressed as mean ± SEM. One-way and two-way analysis of variance was used to compare the differences between three or more groups. Tukey's test was used to compare the differences between the two groups. Differences were statistically indicated significant when p-value was less than .05.

| Cytotoxicity evaluation
MTT and comet assays were used in order to investigate the possible cytotoxic and genotoxic effects of linalool in PC12 cells, after 14 and 18 h exposure. 18 h caused a significant DNA fragmentation to 15.2 ± 1.6% and 21.3 ± 2%, respectively, when compared with the untreated group (5.7 ± 1.6% and 6.2 ± 1.8% DNA in tail).

| Linalool suppressed OGD/R-induced ROS generation in PC12 cells
The results showed that OGD/R was associated with a remarkable increase (328 ± 29.5%, p < .001) in intracellular ROS generation which decreased by (±) linalool ( Figure 3). However, ROS generation was significantly inhibited in the cells pre-incubated with 100 μM of (R)-(−) linalool to 184.3 ± 23.3% (p < .01) following 2 h and to 174.5 ± 25.5% (p < .01) following 6 h. No significant difference was seen between 2 and 6 h pre-incubation of the cells with racemic or

| DISCUSS ION
The main finding of the present study is that linalool significantly improved the PC12 cell survival following ischemia/reoxygenation

F I G U R E 2 Effect of linalool on the viability of ischemic-injured PC12 cells. The cells were pretreated with (±) linalool and (R)-(−) linalool
(1-100 μM;) for 2 (a and c, respectively) and 6 h (b and d, respectively) and then subjected to 12 h of oxygen-glucose-serum deprivation and reoxygenation (OGD/R) condition. The cell viability was determined via the MTT assay. Data were presented as mean ± SEM., ***p < .001, **p < .01 vs. control group, ## p < .001, ### p < .01 vs. OGD/R group. Ischemia is associated with redox imbalance which results in oxidative stress and brain cell apoptosis (Rodríguez-González et al., 2016). It was reported that oxidative stress causes irreversible damage in DNA and other macromolecules (Ge et al., 2018). In the present study, OGD in vitro model was used to mimic the pathological process of ischemia/reperfusion injury, as established previously, and to evaluate the neuroprotective potential of linalool on OGDinduced cell injury.
In line with the previous studies (Khazdair et al., 2015;Yu et al., 2008), we found that excessive amounts of ROS can be generated in PC12 cells under OGD/R condition. Moreover, ROS can promote peroxidation of polyunsaturated fatty acids as indicated by increased malondialdehyde (MDA) production in our study.
Linalool also attenuated benzene-induced oxidative toxicity in rat liver by reduction of advanced oxidation protein products and MDA production, with concomitant elevation of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) contents (Ola & Sofolahan, 2021). Linalool improved renal function following ischemia-reperfusion injury by mitigation of oxidative damage and restoring the activities of anti-oxidant enzymes (Golmohammadi et al., 2021). Linalool also decreased doxorubicin-induced renal F I G U R E 3 Effect of linalool on ROS production in the ischemic-injured PC12 cells. The cells were preincubated with various concentrations of (±) linalool (a) and (R)-(−) linalool (b) for 2 and 6 h and then subjected to 12 h of oxygen-glucoseserum deprivation and reoxygenation (OGD/R) condition. The intracellular ROS was estimated via DCF fluorescence intensity. The cells without any treatment were used as the control. Data were presented as mean ± SEM. ***p < .01 vs. control group, ## p < .01 vs. OGD/R group. In consistent with aforementioned evidences, we observed that linalool dose-dependently suppressed OGD/R-induced ROS and MDA overproductions. As reported in the previous investigations (Jung & Kwak, 2010;Xu et al., 2017a), enhanced cellular antioxidant capacity may be linked to the remarkable difference in the amount of lipid peroxidation between the OGD/R-injured cells pre-incubated with linalool for 2 or 6 h.
Consistently, in a study conducted by Sugawara et al. (1998) it was found that the sedative activity of (±)-linalool could be attributed to (R)-(−) linalool but not (S)-(+)-enantiomer. (R)-(−) Linalool was found to relieve some stress-related physiological parameters of autonomous nervous system (heart rate and blood pressure, etc.). While, (S)-(+)-isoform induced opposite effects including increased heart rate and blood pressure (Höferl et al., 2006). de Sousa et al. (2010) reported elongation of convulsions latency by linalool in animal model of pentylenetetrazol-induced convulsions. However, the anticonvulsant effect of linalool racemate was more potent than its enantiomers. Two enantiomers showed similar qualitative anticonvulsant activities with different potencies.
Subsequent to OGD, ROS activate a cascade of deleterious processes which eventually promote neuronal cell death (Jin et al., 2018). Energy deprivation, mitochondrial dysfunction, and overproduction of free radicals can induce DNA damage which eventually results in cell apoptosis (Guo et al., 2013). Death signals following ischemia-reperfusion injury activate Bcl-2 family F I G U R E 4 Effect of linalool on MDA production in the ischemic-injured PC12 cells. The cells were preincubated with various concentrations of (±) linalool (A) and (R)-(−) linalool (B) for 2 and 6 h and then subjected to 12 h of oxygen-glucoseserum deprivation and reoxygenation (OGD/R) condition. The MDA level was determined via TBARS fluorescence intensity. The cells without any treatment were used as the control. Data were presented as mean ± SEM. ***p < .001 vs. control group, # p < .05, ## p < .01, ### p < .001 vs. OGD/R group. members which leads to activation of the mitochondria-dependent caspase signaling pathway (Mousavi et al., 2010;Shafaei-Bajestani et al., 2014). Bax over-expression can promote the apoptosis (Gao et al., 2016).
Our observations are also in agreement with previous studies revealed the neuroprotective effects of (−) linalool against OGD or H 2 O 2 -stimulated neuronal injury. Linalool protected PC12 cells against H 2 O 2 by attenuation of oxidative stress and apoptosis. Also, linalool inhibited inflammation and oxidative stress in cortical neuronal injury induced by OGD/R (Migheli et al., 2021;Park et al., 2016).
Studies have demonstrated that downregulation of Bcl-2 caused decreased cell viability under ischemia-hypoxia condition. In cerebral ischemia, Bcl-2 was considered as an indicator protein for nerve cell apoptosis (Ouyang & Giffard, 2004;Wei et al., 2005). Since Bcl-2 possesses free radical scavenging activity, it is capable of suppressing peroxidation of lipid and DNA injury exerted by deleterious agents and initiators of apoptosis in PC12 cells (Jang & Surh, 2003).

Previous studies have reported an increase in the expressions of
Bax and cleaved-caspases 3, 9 following cerebral ischemia (Gao et al., 2016;Tian et al., 2018).
As indicated in the previous studies, linalool could reduce rate of cardiomyocyte apoptotic death induced by isoproterenol, by suppressing Bax protein expression (Zheng et al., 2017). Similarly, Gunaseelan et al. (2017) showed that linalool blocked apoptosis of HDFa skin cells following UV-B exposure by modulating of redox status and expressions of Bax and Bcl-2 proteins. In the same way, F I G U R E 5 Effect of linalool on DNA damage in the ischemic-injured PC12 cells. (a and b) representative comet images of the ischemicinjured cells treated with (±) linalool and (R)-(−) linalool. The cells were preincubated with different concentrations (25, 100 μM) of (±) linalool (c) and (R)-(−) linalool (d) for 2 and 6 h and then subjected to 12 h oxygen-glucose-serum deprivation and reoxygenation (OGD/R) condition. Scale bars indicate 0.1 mm. The percent of DNA in the comet tail (% tail DNA) was estimated as an index for the extent of DNA damage. ***p < .001 vs. control group, # p < .05, ### p < .001 vs. OGD/R group.

ACK N OWLED G M ENTS
This study is financially supported by the Vice Chancellery for Research and Technology, Mashhad University of Medical Sciences, Mashhad, Iran (grant numbers: 89551, 901177).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F I G U R E 7
The schematic representation of the mode of actions of (±) linalool and (R)-(−) linalool against PC12 cell death induced by oxygen-glucose-serum deprivation and reoxygenation (OGD/R).